TWI414600B - Photosynthesis bacteria transformant with thermotolerance - Google Patents

Abstract

A photosynthesis bacteria transformant with thermotolerance is disclosed. The transformant includes a plasmid containing gene encoding [FeFe]-hydrogenase. The expression of the gene can enhance the thermotolerance of the photosynthesis bacteria host.

Description

Heat-tolerant photosynthetic hydrogen producing strain

The present invention relates to a photosynthetic hydrogen producing strain, and in particular to a heat-tolerant photosynthetic hydrogen producing strain.

Photosynthetic microorganisms have been widely used in aquaculture, food additive industries or activated sludge for wastewater treatment.

The cultivation cost of photosynthetic microorganisms is relatively high. The main reason is that the fermentation tanks must have a certain degree of illumination (3000-6000 Lux) and appropriate temperature control (30-35 °C) in order to cultivate good quality photosynthetic microorganisms. The most direct way to reduce costs is to use sunlight as a source of light, using sunlight and its accompanying thermal energy to save on the operating cost of the culture tank.

However, when sunlight is used as the photophosphorylation energy source of photosynthetic microorganisms, the temperature of the culture tank is gradually increased due to illumination, which exceeds the temperature range that photosynthetic microorganisms can adapt, thus affecting the physiological state of photosynthetic microorganisms and reducing Cell growth concentration.

In general, to improve the thermal tolerance of microorganisms can be achieved by two methods, one is to express multiple heterologous or homologous chaperone by means of meristem; or in the form of natural mutation Screening microorganisms with heat tolerance traits.

One aspect of the present invention provides a heat-tolerant photosynthetic hydrogen producing strain having a deoxyribonucleic acid fragment encoding a [FeFe]-hydrogenase ([FeFe]-Hydrogenase). body.

According to an embodiment of the invention, the deoxyribonucleic acid fragment encoding the [FeFe]-hydrogenase is derived from the chromosomal DNA of Clostridium thermocellum .

According to an embodiment of the invention, the plastid comprises a sequence of deoxyribonucleic acid fragments as shown in SEQ ID NO: 1. The deoxyribonucleic acid fragment of SEQ ID NO: 1 contains a deoxyribonucleic acid sequence which can be encoded as [FeFe]-hydrogenase. According to an embodiment of the invention, the plastid has a sequence as shown in SEQ ID NO: 3 and is deposited with the Food Industry Development Institute (FIRDI) under the number BCRC 940602.

According to an embodiment of the invention, the photosynthetic hydrogen producing bacteria host is a purple sulphur-free photosynthetic bacterium ( Rhodopseudomonas palustris ).

According to the above, the heat-tolerant photosynthetic hydrogen producing strain of the present invention is used for expressing exogenous [FeFe]-hydrogenase in photosynthetic hydrogen producing bacteria without additional expression of chaperone protein (chaperone protein). ), the host can be adapted to the thermal tolerance of the culture environment of 35 ~ 40 ° C. In particular, the method of the present invention does not use only hydrogenase to enhance the hydrogen production ability of microorganisms, but produces an effect of stabilizing cell physiological activity and maintaining cell growth concentration in a hot environment.

The heat-tolerant photosynthetic hydrogen producing strain of the embodiment of the present invention is different from the optimal growth temperature range of the wild strain at 28-35 ° C, and is more heat-tolerant than the wild-type strain, reducing the limitation of the culture condition. Therefore, the application range of photosynthetic hydrogen-producing bacteria is expanded.

According to an embodiment of the present invention, a plastid containing a dehydroribonucleic acid fragment capable of translating [FeFe]-hydrogenase is transformed into a host of photosynthetic hydrogen producing bacteria, which can improve the adaptability of photosynthetic hydrogen producing bacteria to a hot environment. The transformed photosynthetic hydrogen-producing bacteria can be grown in a culture environment with a temperature of about 40 °C.

As used herein, "adaptation" includes the growth and maintenance of growth of the cells under culture conditions.

Here, the gene encoding the [FeFe]-hydrogenase can be called the hydA gene or the homologous sequence of the hydA gene, which can be obtained from the appropriate strain by means of transfection, or can be artificially synthesized. The way to get this sequence. Strains suitable for use in the present invention to obtain the hydA gene or a sequence homologous to the hydA gene include strains which can survive in the environment of 40-70 ° C, including but not limited to strains of the genus Clostridium . The strain of the genus Clostridium may be, for example, Clostridium thermocellum . Can be obtained through cloning sequence of a gene or a gene homologous to hydA hydA from the chromosomal DNA of the aforementioned strains in; hydA or synthesized gene sequence or by using the synthetic gene with homologous hydA. Those of ordinary skill in the art can use known procedures to obtain the hydA gene or a sequence homologous to the hydA gene without undue experimentation.

The above " hydA gene or a sequence homologous to the hydA gene" includes a sequence represented by SEQ ID NO: 1, a complementary sequence thereof, and a conservative analog, for example, having a degenerate codon substitution ( Degenerative codon substitutions) homologous sequences. Degenerate codon substitutions can be made via a third position in one or more selected codons in the nucleic acid sequence, according to common general knowledge in the art.

In one embodiment of the present invention, a plastid is constructed by using the obtained hydA gene or a sequence homologous to the hydA gene, and a suitable photosynthetic hydrogen producing host cell is transfected with the constructed plastid. The transformed cells transfected with the plastid can translate [FeFe]-hydrogenase to improve the adaptability of the photosynthetic hydrogen-producing host cell to high temperature environment, including adapting to the thermal tolerance of the culture environment around 40 °C.

A plastid suitable for use in the present invention includes, but is not limited to, in one embodiment, the plastid is pMGPHT comprising a deoxyribonucleic acid sequence of SEQ ID NO:3. This plastid contains at least a promoter region and a hydA gene (SEQ ID NO: 1) or a sequence homologous to the hydA gene. The promoter region may comprise a sustained phenotype promoter or an inducible promoter. Unless otherwise specified, the "promoter region" as used herein encompasses a promoter sequence recognized by an RNA polymerase, one or more regulatory sequences recognized by a regulatory gene, and a TATA-BOX sequence. , transcription start point sequence, Shine-Dalgarno (SD) sequence.

The plastids constructed in the manner described above are transfected into appropriate host cells in a well known manner. For example, calcium chloride transformation, electroporation, particle bombardment, and the like.

The photosynthetic hydrogen producing bacteria suitable for use in the present invention may include, but are not limited to, strains of the genus Rhodopseudomonas, the genus Rhodospirillaceae, and the genus Rhodobacter . The strain of Rhodopseudomonas can be, for example, a purple sulphur-free photosynthetic bacterium ( Rhodopseudomonas palustris ).

In addition, the DNA sequence homologous to the hydA gene can be replaced with the preferred codon of the target host according to different hosts to promote the expression of [FeFe]-hydrogenase which is capable of producing hydrogen.

According to an embodiment of the present invention, a fragment of the hydA gene (SEQ ID NO: 2) containing a persistent phenotype promoter region upstream is constructed into a plastid pMGPHT (SEQ ID NO: 3), and transformed into Rhodopseudomonas palustris CGA009 (purchased) From the American Type Culture Collection; ATCC) host.

The preferred embodiments of the present disclosure are described in detail below, examples of which are illustrated in the accompanying drawings.

Example Construction of plastid pMGPHT

The first picture shows the construction flow chart of the plastid pMGPHT. In this example, Clostridium thermocellum ATCC 27405 was used as a template (purchased from the Center for Biological Resources attached to the Food Industry Research), using the introduction of HydA-F (SEQ ID NO: 4) and the introduction of HydA-R (SEQ ID NO: 5). The hydA gene, the start codon was modified to ATG to enhance the expression of the hydA gene in the host; a promoter (Promoter) fragment was added upstream of the hydA gene (SEQ ID NO: 6), and a terminator fragment was added downstream of the hydA gene. (SEQ ID NO: 7), a deoxyribonucleic acid fragment as shown in SEQ ID NO: 2 was obtained, wherein the 5' end has an Xba I cleavage site and the 3' end has a Sac I cleavage site. In this example, the sequence of the promoter and terminator is derived from the pckA gene of R. palustris P4.

The deoxyribonucleic acid fragment of SEQ ID NO: 1 is further ligated with the pMG105 shuttle vector (Shuttle vector; Institute of Research Institute of Innovative Technology for the Earth, Kyoto, Japan; SEQ ID NO: 8), which is cleaved by the same restriction enzyme Xba I/ Sac I. using T ₄ ligase (T ₄ DNA ligase) for engagement in appropriate proportions to obtain the finished pMGPHT plastid construct (SEQ ID NO: 3).

The above plastid colonization operation may include, but is not limited to, the following steps.

In this example, the reactants and reagents of the polymerase chain reaction are carried out in the following ratios: 1 μg of template DNA, 2 μL of dNTP solution (1.25 mM), 2.5 μL of 10 times concentrated polymerase buffer (10X-Taq DNA polymerase buffer), 10 pmole primer pair and 2.5 unit of Taq-polymerase (Taq DNA polymerase), adjusted to a total volume of 25 μL in sterile deionized water, placed in a polymerase chain reaction reactor (GeneAmp PCR system 2400; Perkin Elmer) was subjected to a polymerase chain reaction. The reaction conditions were 94 ° C for 5 minutes, 94 ° C for 1 minute (10 cycles; 10 cycles), 50 ° C for 1 minute, 72 ° C for 1 minute and 20 seconds, and 94 ° C for 1 minute (20 cycles; 20 cycles) The reaction was carried out at 60 ° C for 1 minute, 72 ° C for 1 minute and 20 seconds, and after the reaction was completed, the reaction was continued at 72 ° C for 10 minutes, and then the temperature was lowered to 4 ° C, and the results of the PCR were observed by agarose gel electrophoresis.

The PCR product recovery can be first separated by agarose gel electrophoresis. The colloid containing the target fragment is excised and recovered using methods or commercial kits commonly used in the art for recovering DNA. The recovered product can be subjected to subsequent shearing and joining reactions.

DNA molecular cleavage is performed according to the protocol provided by the restriction enzyme manufacturer. After appropriate DNA sample is mixed with sterile deionized water and restriction enzyme buffer, then add appropriate amount of restriction enzyme to mix evenly, and react at the optimum reaction temperature of the enzyme.

DNA joining reaction is carried cut in accordance with T ₄ ligase manufacturers to provide the process. The plastid DNA and the DNA fragment to be inserted are mixed in an appropriate ratio, mixed with the sterilized deionized water and the ligase buffer, and then mixed with an appropriate amount of ligase, and the reaction is terminated at the optimum reaction temperature of the enzyme for a while. It can be transformed.

Photosynthetic hydrogen production strain transformed strain containing plastid pMGPHT

R. palustris is a purple non-sulfur bacteria (PNSB) that produces hydrogen by nitrogenase. Purple sulfur-free bacteria are photosynthetic microorganisms with the best hydrogen production efficiency, and can produce hydrogen in both light and dark places.

The shuttle plastid pMGPHT was transformed into R. palustris electric competent cells by electric transformation, and colonies with antibiotic resistance of kanamycin were screened after culture. The selected transformed strain was named R. palustris strain pMGPHT.

The above-mentioned transformation plant selection operation may include, but is not limited to, the following steps. For example, in this example, R. palu stris was cultured in Rhodospirillaceae medium under an anaerobic environment at 32 ° C and provided with 50 W/m ² of light. Rhodospirillum medium contains K ₂ HPO ₄ (0.5 g/L), KH ₂ PO ₄ (0.5 g/L), MgSO ₄ ‧7H ₂ O (0.2 g/L), NaCl (0.4 g/L), CaCl ₂ ‧2H ₂ O (0.05 g/L), yeast extract (0.2 g/L), 1.0 g/L Iron citrate solution (5 mL/L) and trace element solution (1 mL). The trace element solution contains ZnCl ₂ (70 mg/L), MnCl ₂ ‧4H ₂ O (100 mg/L), H ₃ BO ₃ (60 mg/L), CoCl ₂ ‧6H ₂ O (200 mg/L), CuCl ₂ ‧2H ₂ O (20 mg/L), NiCl ₂ ‧6H ₂ O (20 mg/L), NaMoO ₄ ‧2H ₂ O (40 mg/L); 25% HCl (1 mg/L).

The above agent may be pre-formulated into a concentrate: (A) KH ₂ PO ₄ + K ₂ HPO ₄ (50 g / L: 50 g / L); (B) MgSO ₄ ‧7H ₂ O (20 g / L); (C) NaCl + CaCl ₂ ‧ 2H ₂ O (40 g / L: 5 g / L). When preparing 300 mL, first fill about 150 mL of deionized water in a triangular flask, then add 3 mL of each concentrate (A) (B) (C), 1.5 mL of ferric citrate (Fe-citrate) solution, trace element solution 0.3 mL, yeast extract (Yeast Extract) 0.06 g, glutamic acid (Glutamate) 0.12 g, and CH ₃ COONa 0.3 g as a carbon source. The carbon source can be replaced with lactic acid (Lactate) as needed. The pH of the medium was adjusted to 7.1 using 0.1 M NaOH, and the top space of the flask was filled with helium to become an anaerobic state.

The solid medium was supplemented with 1.5% bacterial agar (Bacto Agar; Difco) in the liquid medium. After sterilization and dissolution in the autoclave, antibiotics were added according to the experiment, and the culture dish (90×15 mm, Alpha plus) was added. It can be used after it has been coagulated and dried. In this example, the preparation and electroporation transformation of R. palustris electric competent cells was carried out in accordance with the method published by Donohue and Kaplan in 1991. R. palustris was cultured in the above culture solution for 8-24 hours, repeatedly diluted with sterile secondary water, centrifuged 7-8 times, and then 10% glycerol was added to prepare an electric competent cell. After mixing 40 μL of polyethylene glycol 6000 (PEG 6000) (50%), 20 μL of plastids and 40 μL of electric competent cells, the shock wave was shocked by 8.5 m/sec and placed on ice for 1 minute. (BIO-RAD GENE PULSER II), electrical transformation conditions are resistance 400Ω, 2.5kV, capacitance 25μF, electric field strength 12.5kV/cm. The electrotransformed cells were cultured in a medium containing Kanamycin (200 μg/mL), and transformed strains having antibiotic resistance were selected.

Fig. 2 is a result of gel electrophoresis after screening the transformed transformant R. palustris strain pMGPHT plastid and digesting with restriction enzyme Bgl II. Among them, the lane M is a molecular weight marker, and the lane 1 is the result of the extracted plastid by Bgl II. According to the pMGPHT restriction enzyme map shown in Fig. 1, after cleavage with Bgl II, a fragment of 5704 base pairs (bp), 1460 bp, and 471 bp was theoretically obtained. The gel electrophoresis results in Figure 2 showed fragments of about 5.7 kb, about 1.5 kb, and about 0.47 kb, which were consistent with the estimated fragment size and confirmed to be pMGPHT.

In addition, the colony of the completed R. palustris strain pMGPHT retained kanamycin antibiotic resistance after the second passage, indicating that the plastid pMGPHT is stable in R. palustris . Figure 3 shows the results of detecting the hydA gene of pMGPHT in the host by reverse transcription polymerase chain reaction (RT-PCR).

The total RNA of the transformed strain R. palustris strain pMGPHT was extracted and extracted with the specificity of the hydA sequence PHT-45F(R) (SEQ ID NO: 9) in combination with PHT-45F(L) ( SEQ ID NO: 10) RT-PCR was performed to detect whether the selected hydA gene was successfully expressed in the host. M is the molecular weight marker (100 bp DNA Ladder), RT (+) is the experimental group (adding reverse transcriptase and reaction buffer, primer PHT-45F (R), total RNA), RT (-) is the control group (not Add reverse transcriptase). Shown by the results of FIG. 3, RT (+) experiment the DNA complementary to the successful growth of hydA (Complementary DNA; cDNA) fragments, indeed it represents hydA gene expression in the host.

Effect of exogenous [FeFe]-hydrogenase protein expression on enhancing host heat tolerance

Figure 4 is a comparison of the cell growth rates of the R. palustris strain pMGPHT transformed strain and the control group containing the empty vector (pMG105; no hydA gene) at different temperature conditions.

In the temperature test, the strains to be tested were respectively cultured in a water bath at 38 ° C, 40 ° C, 42 ° C, and 45 ° C in an anaerobic sealed test tube. The cell growth condition (concentration) was detected by a spectrophotometer. Each set of data is expressed as the mean and standard deviation of at least three independent experiments.

The results in Fig. 4 show that the R. palustris strain pMGPHT transformed strain has a significantly shorter cell doubling time than the control group containing only empty vector when cultured above 40 °C.

Among them, as shown in the following Table 1, when cultured at 38 ° C, 40 ° C, 42 ° C and 45 ° C, R. palustris strain pMGPHT transformed strain can shorten the ratio of cell doubling time (control group / transformation strain) compared with the control group The values of 1.04, 1.46, 1.32 and 1.34, respectively, indicate that the R. palustris strain pMGPHT transformant does have the ability to adapt to higher temperature culture environments.

Figure 5 is a schematic diagram of the mechanism of exogenous [FeFe]-hydrogenase for enhancing the heat tolerance of photosynthetic hydrogen-producing bacteria.

Photosynthetic microorganisms use Nitrogenase to produce hydrogen to balance the reducing energy in cells. By consuming adenosine triphosphate (ATP), hydrogen protons are converted to hydrogen, which requires higher activation energy. Its chemical action is:

2H ⁺ +2e ^- +4ATP → H ₂ +4ADP+4Pi

Hydrogenase can decompose organic matter in an anaerobic fermentation environment, and some electrons generated by the process of metabolizing organic matter are generated by hydrogen transfer to hydrogen protons in water. Its chemical action is:

2H ⁺ +2e ^- → H ₂

As shown in the route (A) of Figure 5, when photosynthetic hydrogen-producing bacteria use hydrogenase to produce hydrogen, it is necessary to consume ATP to convert hydrogen protons into hydrogen. However, when the cells additionally express exogenous [FeFe]-hydrogenase, it is speculated that it can provide another energy-saving intracellular reducing energy balance pathway (B).

When the cells are cultured in a harsh environment, they may affect cell metabolism and slow down the growth rate. When the extracellular [FeFe]-hydrogenase is additionally expressed by the cells, hydrogen can be produced partially via the route (B), and the energy consumed by the route (A) is relatively saved, thereby saving the energy by the machine to promote the bacteria. Body growth helps to adapt to a higher temperature culture environment.

As can be seen from the above-described embodiments of the present invention, the present invention differs from the conventional meristem method in that a host protein is protected in a manner of expressing a plurality of protein protectors to enhance the heat tolerance of the microorganism. The photosynthetic hydrogen producing strain of the embodiment of the invention can effectively enhance the heat tolerance of the photosynthetic hydrogen producing bacteria while expressing the heterologous [FeFe]-hydrogenase, so that the original can only adapt to 28-35 ° C Photosynthetic hydrogen-producing bacteria grown under the environment can also adapt to temperatures around 40 °C.

Therefore, the photosynthetic hydrogen producing strain having the exogenous [FeFe]-hydrogenase of the present invention can be adapted to the problem of an increase in the temperature of the culture tank after the sunlight irradiation. By applying the photosynthetic hydrogen producing strain of the present invention, the sunlight and the source of light and heat energy of the photosynthetic bacteria can be achieved, thereby saving the operation cost of the culture tank. Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention, and the present invention can be modified and modified without departing from the spirit and scope of the present invention. The scope is subject to the definition of the scope of the patent application attached.

<110>National Chung Hsing University

<120>Heat-tolerant photosynthetic hydrogen producing strain

<160>9

<210>SEQ ID NO: 1

<211>1739

<212>DNA

<213>Artificial sequence

<220>CDS

<223> hydA gene sequence

<400>1

<210>SEQ ID NO: 2

<211>1894

<212>DNA

<213>Artificial sequence

<220>CDS

<223> DNA fragment capable of transcribed [FeFe]-Hydrogenase

<400>2

<210>SEQ ID NO: 3

<211>7635

<212>DNA

<213>Artificial sequence

<220>cyclic nucleic acid sequence

<223> plastid pMGPHT sequence which expresses HydA protein in a photosynthetic bacteria host

<400>3

<210>SEQ ID NO: 4

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223> Increment of the hydA gene (HydA-F)

<400>4

<210>SEQ ID NO: 5

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223> Increment of the hydA gene (HydA-R)

<400>5

<210>SEQ ID NO: 6

<211>124

<212>DNA

<213>Artificial sequence

<220>promoter

<223> The pckA gene derived from Rhodopseudomonas palustris P4

<400>6

<210>SEQ ID NO: 7

<211>52

<212>DNA

<213>Artificial sequence

<220>terminater

<223> The pckA gene derived from Rhodopseudomonas palustris P4

<400>7

<210>SEQ ID NO: 8

<211>7635

<212>DNA

<213>Artificial sequence

<220>cyclic nucleic acid sequence

<223> Shuttle carrier pMG105

<400>8

<210>SEQ ID NO: 9

<211>27

<212>DNA

<213>Artificial sequence

<220>primer

<223>Introduction to the hydA sequence PHT-45F(R)

<400>

<210>SEQ ID NO: 10

<211>28

<212>DNA

<213>Artificial sequence

<220>primer

<223>Specific primer for the hydA sequence PHT-45F(L)

<400>

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

The first figure is a flow chart of the construction of the plastid pMGPHT.

Fig. 2 Transformation strain R. palustris strain pMGPHT plastids were confirmed by gel electrophoresis after restriction enzyme cleavage.

Figure 3 shows the sequence number of pMGPHT detected by reverse transcription polymerase chain reaction: 2 The results of fragment expression in the host.

Figure 4 is a comparison of cell growth rates of R. palustris strain pMGPHT transformed strains and control groups at different temperature conditions.

Figure 5 is a schematic diagram of the mechanism of exogenous [FeFe]-hydrogenase for enhancing the heat tolerance of photosynthetic hydrogen-producing bacteria.

Claims (1)

A heat-tolerant photosynthetic hydrogen producing strain transformed strain comprising a sequence of 3: deoxyribonucleic acid sequence deposited in the Food Industry Development Institute (FIRDI), number BCRC 910506.